Hi, I am phd student and I am working on plankton specie called Pseudo-nitzschia. I sent my first enviromental samples (filtered from sea-water) to Novogen for Ilumina sequencing and got some bar results. 50 samples did not pass quality check (out of 107). Intresting thing is that my samples are sent in triplicates, and in some samples 2 out of 3 replicates passed quality check and one did not. I dont have any spare samples, these were collected over period of two years. Also I designed my primers based on Sanger library I made. Also I checked concentration of DNA on Qubit and it was really good (but these are enviromental samples so we expected that.
Is there anyone who proceeded sequencing with fail quality check results? Thanks !
Did the company mention why those failed and they failed at what point? Not enough DNA? Did not amplify? Did not produce libraries?
It would be helpful to know:
(1) Did you prepare and barcode the sequencing library yourself?
(2) What methods of library prep did you use?
(3) Did you include QC checks in between steps?
My best guess without the benefit of any of this information is that your primers might not be very efficient and are generating only a very low concentration amplicon after one or both PCR reactions.
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